Picornaviruses are non-enveloped viruses and cannot rely on fusion of a viral membrane with a host cell membrane to gain access to the cytoplasm. They rely on the ability of capsid proteins to undergo as-yet-uncharacterized conformational changes for the viral RNA to breach the host-membrane barrier. These irreversible changes include externalization of peptides from the interior of the capsid shell. Poliovirus is the prototypical picornavirus and serves as an excellent model system. Our broad aim is to gain knowledge of viral structural dynamics associated with host-membrane attachment and genome release by using electron microscopy and three-dimensional data analysis (3DEM). The picornaviruses share a high degree of structural homology with one another and, therefore, the models and methodologies developed during this study will be relevant to other picornaviruses. The two specific aims of this proposed study are 1) to determine the 3DEM structures of poliovirus intermediates during genome-release, and 2) to determine 3DEM structures of such intermediates attached to liposomes. The experimental steps to be carried out are listed below: a. Optimize conditions for preparing vitrified EM grids enriched with poliovirus particles at a particular stage (early or late) of RNA-release. b. Collect EM data (both un-tilted and tomographic) using the Polara and Tecnai F30 microscopes. c. Determine both icosahedral and asymmetric 3D reconstructions of RNA-release intermediates from the 2D EM images using the program PFT for un-tilted data and IMOD for tomograms. d. Build pseudo-atomic models to fit the higher-resolution EM maps, to help account for density changes. PUBLIC HEALTH RELEVANCE: The Picornavirus family includes cardioviruses, foot-and-mouth disease virus, rhinoviruses, hepatitis A virus and enteroviruses. Among these, there are significant causes of human and veterinary diseases. Detailed knowledge of the molecular mechanisms that these viruses use for membrane attachment and genome release would help in the development of antiviral strategies to block the earliest stages of infection and reduce pathogenicity.